A Water-Based Biocoating to Increase the Infection Resistance and Osteoconductivity of Titanium Surfaces.

As the population ages, the number of patients undergoing total hip arthroplasty (THA) and total knee arthroplasty (TKA) continues to increase. Infections after primary arthroplasty are rare but have high rates of morbidity and mortality, as well as enormous financial implications for healthcare systems. Numerous methods including the use of superhydrophobic coatings, the incorporation of antibacterial agents, and the application of topographical treatments have been developed to reduce bacterial attachment to medical devices. However, most of these methods require complex manufacturing processes. Thus, the main purpose of this study was to apply biocoatings to titanium (Ti) surfaces to increase their infection resistance and osteoconductivity via simple processes, without organic reagents. We modified titanium surfaces with a combination of aminomalononitrile (AMN) and an antibiotic-loaded mesoporous bioactive glass (MBG) and evaluated both the antibacterial effects of the coating layer and its effect on osteoblast proliferation and differentiation. The properties of the modified surface, such as the hydrophilicity, roughness, and surface morphology, were characterized via contact angle measurements, atomic force microscopy, and scanning electron microscopy. The cell proliferation reagent WST-1 assay and the alkaline phosphatase (ALP) assay were used to determine the degrees of adhesion and differentiation, respectively, of the MG-63 osteoblast-like cells on the surface. Antimicrobial activity was evaluated by examining the survival rate and inhibition zone of Escherichia coli (E. coli). The AMN coating layer reduced the water contact angle (WCA) of the titanium surface from 87° ± 2.5° to 53° ± 2.3° and this change was retained even after immersion in deionized water for five weeks, demonstrating the stability of the AMN coating. Compared with nontreated titanium and polydopamine (PDA) coating layers, the AMN surface coating increased MG-63 cell attachment, spreading, and early ALP expression; reduced E. coli adhesion; and increased the percentage of dead bacteria. In addition, the AMN coating served as an adhesion layer for the subsequent deposition of MBG-containing antibiotic nanoparticles. The synergistic effects of the AMN layer and antibiotics released from the MBG resulted in an obvious E. coli inhibition zone that was not observed in the nontreated titanium group.

The influence of laser intensity activated plasmonic gold nanoparticle-generated photothermal effects on cellular morphology and viability: a real-time, long-term tracking and monitoring system.

In this study, a microfluidic apparatus embedded with microstructures was designed and aligned with a laser and dark-field microscope for real-time, long-term observation of photothermal effects on cells. Gold nanorods (AuNRs, 10 ppm) were incubated with MG-63 human osteosarcoma cells for 3 h. Then, the cells were exposed to a continuous-wave laser at a wavelength of 830 nm for 10, 20, and 30 min at 5, 9, 14, 24, and 32 W cm-2. Subsequent changes in morphology were observed. Under different conditions, cell membrane blebbing occurred at different times, indicating that actin filaments were destroyed in large quantities and apoptosis was induced. In suitable conditions, we first induced slight cell injury by causing cytoskeletal fractures with a high-energy laser; then, the cells were irradiated with a low-energy laser at 0.3 W cm-2. We found that among cells treated with the high-energy laser, cells treated additionally with a low-energy laser showed extended viability compared with cells that did not receive the additional treatment.

Selective Targeting and Restrictive Damage for Nonspecific Cells by Pulsed Laser-Activated Hyaluronan-Gold Nanoparticles.

Herein, we describe an approach that immobilizes low-molecular-weight hyaluronic acid (low-MW HA) on the surface of gold nanoparticles (GNPs), which can serve as a cellular probe and photodamage media, to evaluate the selectivity and efficiency of HA-based GNPs (HGNPs) as a mediator of laser-induced photothermal cell damage. In addition, it is known that solid tumors contain a higher content of low-MW HA than normal tissues. Thus, we used low-MW HA rather than high-MW HA used in other studies. In the present study, we conjugated low-MW HA, which is a linear polysaccharide with a disaccharide repeat unit, to prevent a reduction of the ligand-receptor binding efficiency in contrast to the conjugation of protein or peptides, which have unique three-dimensional structures. Three cell lines-MDA-MB-435 S (with CD44), MDA-MB-453 and NIH/3T3 (both are without CD44)-were investigated in the study, and qualitative observations were conducted by dark-field microscopy and laser scanning confocal microscopy (LSCM). In addition, quantitative measurements calculated using inductively coupled plasma emissions were taken for comparison. Our results showed that within the same treatment time, the uptake dosage of HGNPs by the MDA-MB-435 S cells was higher than that by the MDA-MB-453 and NIH 3T3 cells. Meanwhile, HGNPs uptake by the untreated MDA-MB-435 S cells was higher than that of MDA-MB-435 S cells with CD44 blocked by antibodies or silencing CD44 expression. This result implies that receptor-mediated endocytosis can enhance the cellular uptake of HGNPs. In addition, when exposed to a low-power pulsed laser, the former cell morphologies showed a more laser-induced giant plasma membrane vesicles (GPMV) than the latter morphologies. Therefore, this study utilized the specific photothermal property of HA-modified GNPs with laser-induced blebs to create a possible new method for medical applications.

A pneumatically-driven microfluidic system for size-tunable generation of uniform cell-encapsulating collagen microbeads with the ultrastructure similar to native collagen.

This study reports a microfluidic system for high throughput, uniform, and size-tunable generation of cell-containing collagen microbeads. The principle is based on two pneumatically-driven mechanisms to achieve multi-channel mixture suspension transportation, and to actuate the spotting actions of micro-vibrators that continuously generate tiny collagen micro-droplets into a thin oil layer and then a sterile Pluronic® F127 surfactant solution located below. The temporarily formed collagen microdroplets are then thermally gelatinized. By regulating the feeding rate of cells/collagen suspension, and the spotting frequency of micro-vibrator, the size of the collagen microbeads can be manipulated. One of the key technical features is its capability to generate uniform collagen microbeads (coefficient of variation: 5.4-8.6 %) with sizes ranging from 73.9 to 349.3 µm in diameter. This is currently difficult to achieve using the existing methods particularly the generation of cell-encapsulating collagen microbeads with diameter less than 100 µm. Another advantageous trait is that the ultrastructure of the generated collagen microbeads is similar to that found in native collagen. In this study, moreover, the use of the proposed device for the microencapsulation of 3T3 cells in collagen microbeads has been successfully demonstrated showing that the encapsulated cells maintained high cell viability (96 ± 2 %). Furthermore, a reasonable proliferative capability of the encapsulated cells was observed during 7 days culture. As a whole, the proposed device has opened up a new route to generate cell-containing collagen microbeads, which is found particularly meaningful for biomedical applications.

Preparation and Evaluation of Aminomalononitrile-Coated Ca-Sr Metal-Organic Frameworks as Drug Delivery Carriers for Antibacterial Applications.

After orthopedic surgery, antibiotics are usually employed to reduce the risk of infection. If it is possible to enhance antimicrobial functionality and incorporate antimicrobial agents into the bone-filling matrix, not only it can promote bone tissue regeneration, but it can also enable localized administration of medication to elevate antibacterial efficacy. Meanwhile, previous studies have shown that calcium and strontium can support the growth of osteoblastic cells and diminish bone resorption or deterioration. In the past few years, metal-organic frameworks (MOFs) have been widely used as drug carriers owing to their characteristic advantages. In this study, a MOF was prepared in an aqueous solution by a simple coprecipitation method with the organic ligand 1,3,5-tricarboxylic benzene (H3BTC) as a linker to form Ca-Sr-MOF. Furthermore, the Ca-Sr-MOF was coated with aminomalononitrile (AMN), which adhered through the electrostatic interactions between H3BTC and AMN. With this MOF (Ca-Sr-AMN-MOF), AMN polymerization reactions can occur in aqueous environments, and a polymer layer was observed on the MOF surface with moderate hydrophilicity. The prepared Ca-Sr-MOF and Ca-Sr-AMN-MOF were characterized by Fourier transform infrared spectroscopy, powder X-ray diffraction, scanning electron microscopy, high-resolution transmission electron microscopy, and UV-visible spectroscopy. Finally, tetracycline (TC) was selected as the model drug to measure the drug loading efficiency, release profile, and antibiotic activity. The percent cumulative drug release of TC from Ca-Sr-MOF and Ca-Sr-AMN-MOF was 55.15 and 9.1%, respectively. The antibacterial effectiveness of TC-loaded MOF against Gram-negative Escherichia coli bacteria was evaluated, revealing the remarkable antimicrobial performance of these substances.

Gold-Nanoparticles-Enhanced Production of Reactive Oxygen Species in Cells at Spread-Out Bragg Peak under Proton Beam Radiation.

This study investigates the radiobiological effects of gold nanoparticles (GNPs) as radiosensitizers for proton beam therapy (PBT). Specifically, we explore the enhanced production of reactive oxygen species (ROS) in GNP-loaded tumor cells irradiated by a 230 MeV proton beam in a spread-out Bragg peak (SOBP) zone obtained by a passive scattering system. Our findings indicate that the radiosensitization enhancement factor is 1.24 at 30% cell survival fraction, 8 days after 6 Gy proton beam irradiation. Since protons deposit the majority of their energy at the SOBP region and interact with GNPs to induce more ejected electrons from the high-Z GNPs, these ejected electrons then react with water molecules to produce excessive ROS that can damage cellular organelles. Laser scanning confocal microscopy reveals the excessive ROS induced inside the GNP-loaded cells immediately after proton irradiation. Furthermore, the damage to cytoskeletons and mitochondrial dysfunction in GNP-loaded cells caused by the induced ROS becomes significantly severe, 48 h after proton irradiation. Our biological evidence suggests that the cytotoxicity of GNP-enhanced ROS production has the potential to increase the tumoricidal efficacy of PBT.

Tunable micropatterned substrates based on poly(dopamine) deposition via microcontact printing.

A simple technique was developed to fabricate tunable micropatterned substrates based on mussel-inspired surface modification. Polydopamine (PDA) was developed on polydimethylsiloxane (PDMS) stamps and was easily imprinted to several substrates such as glass, silicon, gold, polystyrene, and poly(ethylene glycol) via microcontact printing. The imprinted PDA retained its unique reactivity and could modulate the chemical properties of micropatterns via secondary reactions, which was illustrated in this study. PDA patterns imprinted onto a cytophobic and nonfouling substrates were used to form patterns of cells or proteins. PDA imprints reacted with nucleophilic amines or thiols to conjugate molecules such as poly(ethylene glycol) for creating nonfouling area. Gold nanoparticles were immobilized onto PDA-stamped area. The reductive ability of PDA transformed silver ions to elemental metals as an electroless process of metallization. This facile and economic technique provides a powerful tool for development of a functional patterned substrate for various applications.

Gold Nanoparticles Enhancing Generation of ROS for Cs-137 Radiotherapy.

Radiotherapy is an important modality for the treatment of cancer, e.g., X-ray, Cs-137 γ-ray (peak energy: 662 keV). An important therapy pathway of radiation is to generate the double strand breaks of DNA to prohibit the proliferation of cancer cells. In addition, the excessive amount of reactive oxygen species (ROS) is induced to damage the organelles, which can cause cellular apoptosis or necrosis. Gold nanoparticles (GNPs) have been proven potential as a radiosensitizer due to the high biocompatibility, the low cytotoxicity and the high-Z property (Z = 79) of gold. The latter property may allow GNPs to induce more secondary electrons for generating ROS in cells as irradiated by high-energy photons. In this paper, the radiobiological effects on A431 cells with uptake of 55-nm GNPs were studied to investigate the GNPs-enhanced production of ROS on these cells as irradiated by Cs-137 γ-ray. The fluorescence-labeling image of laser scanning confocal microscopy (LSCM) shows the excessive expression of ROS in these GNPs-uptake cells after irradiation. And then, the follow-up disruption of cytoskeletons and dysfunction of mitochondria caused by the induced ROS are observed. From the curves of cell survival fraction versus the radiation dose, the radiosensitization enhancement factor of GNPs is 1.29 at a survival fraction of 30%. This demonstrates that the tumoricidal efficacy of Cs-137 radiation can be significantly raised by GNPs. Because of facilitating the production of excessive ROS to damage tumor cells, GNPs are proven to be a prospective radiosensitizer for radiotherapy, particularly for the treatment of certain radioresistant tumor cells. Through this pathway, the tumoricidal efficacy of radiotherapy can be raised.

Influence of the Components and Orientation of Hydroxyapatite Fibrous Substrates on Osteoblast Behavior.

Synthetic hydroxyapatite has good biocompatibility, bioactivity and osteoconductive ability because its chemical properties and biological properties are similar to those of bioapatite in bone tissue. Strontium-substituted hydroxyapatite has better degradability than hydroxyapatite and can both promote osteogenesis and inhibit adipogenesis in mesenchymal stem cells. Hence, hydroxyapatite and strontium-substituted hydroxyapatite are widely used as bone graft materials, cell carriers and drug/gene delivery carriers. In addition, osteoblasts cultured on aligned nanofibrous substrates had higher expression of osteogenesis-related genes than did those cultured on random nanofibrous substrates. However, to date, no study has explored the effects of the components and orientation of hydroxyapatite nanofibrous substrates on osteoblastic behavior. In this study, a random hydroxyapatite nanofibrous substrate (R-HANF), a random strontium-substituted hydroxyapatite nanofibrous substrate (R-SrHANF), an aligned hydroxyapatite nanofibrous substrate (A-HANF) and an aligned strontium-substituted hydroxyapatite nanofibrous substrate (A-SrHANF) were successfully fabricated by using the electrospinning technique. The effect of fiber composition on osteoblast-like MG63 cells was assessed by evaluating cell morphology, cell proliferation and osteogenesis-related gene expression. The results showed that MG63 cells cultured on A-SrHANF had higher osteogenesis-related gene expression than those cultured on A-HANF. Additionally, MG63 cells were cultured on R-SrHANF and A-SrHANF to evaluate the effects of fiber orientation on cell behavior. On A-SrHANF, the cells aligned along the direction of the nanofibers, with typical bipolar morphologies, and exhibited higher osteogenesis-related gene expression than cells on R-SrHANF. Hence, the components and orientation of hydroxyapatite nanofibrous substrates are critical parameters affecting the osteogenesis process.

Development of high-throughput perfusion-based microbioreactor platform capable of providing tunable dynamic tensile loading to cells and its application for the study of bovine articular chondrocytes.

Mammalian cells are sensitive to extracellular microenvironments. In order to precisely explore the physiological responses of cells to tensile loading, a stable and well-defined culture condition is required. In this study, a high-throughput perfusion-based microbioreactor platform capable of providing dynamic equibiaxial tensile loading to the cultured cells under a steady culture condition was proposed. The mechanism of generating tensile stimulation to cells is based on the pneumatically-driven deformation of an elastic polydimethylsiloxan (PDMS) membrane which exerts tensile loading to the attached cells. By modulating the magnitude and frequency of the applied pneumatic pressure, various tensile loading can be generated in a controllable manner. In this study, the microbioreactor platform was designed with the aid of the experimentally-validated finite element (FE) analysis to ensure the loading of tensile strain to cells is uniform and definable. Based on this design, the quantitative relationship between the applied pneumatic pressure and the generated tensile strain on the PDMS membrane was established via FE analysis. Results demonstrated that the proposed device was able to generate the tensile strain range (0~0.12), which covers the physiological condition that articular chondrocytes experience tensile strain under human walking condition. In this study, moreover, the effect of tensile loading on the metabolic, biosynthetic and proliferation activities of articular chondrocytes was investigated. Results disclosed that the dynamic tensile loading of 0.12 strain at 1 Hz might significantly up-regulate the synthesis of glycosaminoglycans while such stimulation was found no significant influence on the metabolic activity, the synthesis of collagen, and the proliferation of chondrocytes. Overall, this study has presented a high throughput perfusion micro cell culture device that is suitable for precisely exploring the effect of tensile loading on cell physiology.

The Effect of Quasi-Spherical Gold Nanoparticles on Two-Photon Induced Reactive Oxygen Species for Cell Damage.

The performance of quasi-spherical gold nanoparticles (GNPs) on the generation of reactive oxygen species (ROS) to cause cell damage, as irradiated by a two-photon laser, is studied. In this mechanism, hot electrons are generated from GNPs as irradiated by the two-photon laser, reacting with the molecules in the medium to produce ROS. We used laser scanning confocal microscopy with a low-fluence femtosecond Ti:Sapphire laser of 800 nm to observe the generated ROS in A431 cells, which were incubated with GNPs in advance. Subsequently, the cell morphology, cytoskeleton, and viability were investigated. In comparison with the control (no GNPs), the expression of ROS in these GNP-treated cells was enhanced after irradiation by the two-photon laser. Additionally, the disruption of cytoskeletons and the follow-up apoptosis of these GNP-treated cells are significantly increased as the number of laser shots increases. Moreover, we used N-acetyl-L-cysteine (NAC), an antioxidant, to inhibit the formation of ROS, to clarify whether the cytoskeletal disruption is caused by ROS rather than photothermal effects. Our results show that after two-photon irradiation, the ROS expression in these cells treated with GNPs plus NAC was significantly reduced. In addition, the cytoskeletal damage of these cells treated with GNPs and NAC was less than that of those treated with GNPs but without NAC; their cell viability after three days was almost the same with the control. These results illustrate that the induced ROS from the two-photon excited GNPs is the main cause of cell damage. The study may pave a way for the use of GNPs as a photosensitized therapeutic agent for two-photon photodynamic therapy on tumor treatment.

The Effect of Strontium-Substituted Hydroxyapatite Nanofibrous Matrix on Osteoblast Proliferation and Differentiation.

Natural bone tissue consists primarily of bioapatite and collagen. Synthetic hydroxyapatite (HA) possesses good biocompatibility, bioactivity, and osteoconductivity due to its chemical and biological similarity to bioapatite. Hence, HA has been widely used as a bone graft, cell carrier and drug/gene delivery carrier. Moreover, strontium-substituted hydroxyapatite (SrHA) can enhance osteogenic differentiation and inhibit adipogenic differentiation of mesenchymal stem cells. Hence, SrHA has the potential to be used as a bone graft for bone regeneration. It is widely accepted that cell adhesion and most cellular activities are sensitive to the topography and molecular composition of the matrix. Electrospun polymer or polymer-bioceramic composite nanofibers have been demonstrated to enhance osteoblast differentiation. However, to date, no studies have investigated the effect of nanofibrous bioceramic matrices on osteoblasts. In this study, hydroxyapatite nanofiber (HANF) and strontium-substituted hydroxyapatite nanofiber (SrHANF) matrices were fabricated by electrospinning. The effect of the HANF components on MG63 osteoblast-like cells was evaluated by cell morphology, proliferation, alkaline phosphatase activity (ALP) and gene expression levels of RUNX2, COLI, OCN and BSP. The results showed that MG63 osteoblast-like cells exhibited higher ALP and gene expression levels of RUNX2, COLI, BSP and OCN on the SrHANF matrix than the HANF matrix. Hence, SrHANFs could enhance the differentiation of MG63 osteoblast-like cells.

An aqueous-based process to bioactivate poly(ε-caprolactone)/mesoporous bioglass composite surfaces by prebiotic chemistry-inspired polymer coatings for biomedical applications.

Despite the wide use of aliphatic polyesters, such as poly(L-lactic acid) (PLLA) and poly(ε-caprolactone) (PCL), for many biomedical applications, these materials are limited due to their hydrophobic properties and lack of functional groups to bond with ligands to enhance the cell reorganization. Recently, a composite consisting of bioglass and PCL was demonstrated to enhance the mechanical strength and to improve the degradation rate. Although numerous approaches have been developed to improve the wettability of aliphatic polyesters to create a favorable interface with cells, only few surface modification methods can be independently applied to surfaces with different material. In this work, mesoporous bioglass (MBG) nanoparticles embedded in PCL films were modified by the polymerization of aminomalonitrile (AMN) with 3,4,5-trihydroxybenzaldehyde (THBA). The copolymer layer was further utilized as a mediator to conjugate chitosan and evaluate the antibacterial efficacy. Our results show that the hydrophilicity of the composite membranes significantly improved after treatment. In addition, after immersion in simulated body fluid (SBF) for 14 days, hydroxyapatite formation was only observed on the treated membranes. This result demonstrates that the surface treatment did not alter the MBG bioactivity. Moreover, the cell culture results reveal that the extension level of cells and expression of alkaline phosphatase activity (ALP) of osteoblast-like (MG63) cells were higher on treated composite films compared to untreated ones. The results imply that the treatment procedure can be simultaneously and homogeneously applied to the organic/inorganic composites. In addition, Staphylococcus aureus adhesion on AMN-co-THBA and chitosan/ AMN-co-THBA was significantly lower than untreated PCL. Moreover, the percentage of dead bacteria was highest on the chitosan/ AMN-co-THBA membranes. These results indicate that the AMN-co-THBA modification can be used in composite materials and complex constructs, and it provides a potential method to create versatile surface properties for biomedical applications.

Single-photon and two-photon cellular imagings of gold nanorods and dyes.

A method to obtain the expressions of gold nanorods (GNRs) and dye molecules simultaneously is proposed for the single-photon and two-photon cellular imagings by using laser scanning confocal microscopy. For our experiment, GNRs with an average aspect ratio of 2.14 were synthesized using electrochemical method, and the peak of absorption spectrum of GNRs is at 600 nm. The human breast cancer cell lines (MDA-MB-435) were studied by incubating them with GNRs for 20 hours and then staining their nuclei with dye molecules-Prodium Iodide (PI). For the single-photon imaging, different CW lasers (458, 488, 514, 561, and 633 nm) were used individually to irradiate the samples. By adjusting the ranges of two bandpass filters for the detection, the scattered light from the GNRs due to surface plasmon resonance (SPR) and the fluorescence from PI can be induced simultaneously but be detected separately without crosstalk. Furthermore, the two cellular images can be merged together to become a composited cellular image. The TEM image shows that several clusters of GNRs internalized by the vesicles are distributed sparsely inside the cytoplasm, due to the endocytosis of the cells. The aggregation of GNRs causes SPR band broadened. Therefore strong scattered light from GNRs can almost be induced by different-wavelength lasers irradiating. However, the expression of PI can only be detected by the exciting lasers with a wavelength shorter than 600 nm. For the two-photon imaging of these cells internalizing GNRs, an ultrafast Ti:Sapphire IR-laser (800 nm) was used for irradiating the sample, and two bandpass filters were also adjusted to distinguish the photoluminescence of GNRs from the fluorescence of PI.

Gelatin Hydrogels Reinforced by Absorbable Nanoparticles and Fibrils Cured In Situ by Visible Light for Tissue Adhesive Applications.

Most gelatin hydrogels used in regenerative medicine applications today are fabricated by photocrosslinking due to the convenience and speed of this method. However, in most cases photoinitiators are used, which require UV light, which, in turn, can cause cell and tissue damage, or using functionalized gelatin. Recently, ruthenium (II) tris-bipyridyl chloride has been studied as an initiator that can induce dityrosine bond formation using visible light. In addition, continuous fibrils and small particles are often used to reinforce composite materials. Therefore, this study investigated the visible-light-induced photocrosslinking of native gelatin molecules via dityrosine bonds formation as well as gel reinforcement by collagen fibrils and mesoporous bioactive glass (MBG) particles. The results show that collagen and MBG exerted a synergistic effect on maintaining gel integrity with a dental LED curing light when the irradiation time was shortened to 30 s. Without the two reinforcing components, the gel could not form a geometric shape stable gel even when the exposure time was 120 s. The shear strength increased by 62% with the collagen and MBG compared with the blank control. Furthermore, our results demonstrate that the addition of collagen and MBG enhanced gel stability in an artificial saliva solution. These results demonstrate the considerable advantages of using tyrosine-containing biomolecules, and using a dental LED curing light for the crosslinking of hydrogels in terms of their suitability and feasibility for use as bioadhesives in confined clinical working space, such as the oral cavity, and in application as in situ-crosslinked injectable hydrogels.

Collagen Scaffolds Containing Hydroxyapatite-CaO Fiber Fragments for Bone Tissue Engineering.

Collagen (COL) and hydroxyapatite (HAp) are the major components of bone, therefore, COL-HAp composites have been widely used as bone substitutes to promote bone regeneration. We have reported that HAp-CaO fibers (HANFs), which were fabricated by a sol-gel route followed by an electrospinning technique, possessed good drug-loading efficiency and limited the burst release of tetracycline. In the present study, we used HANF fragments to evaluate the effects of COL-HANF scaffolds on MG63 osteoblast-like cell behaviors. COL-HANF composite scaffolds in which the average diameter of HANFs was approximately 461 ± 186 nm were fabricated by a freeze-drying process. The alkaline phosphatase activity and the protein expression levels of OCN and BSP showed that compared with COL alone, the COL-HANF scaffold promoted the differentiation of MG63 osteoblast-like cells. In addition, the bone regeneration ability of the COL-HANF scaffold was examined by using a rabbit condylar defect model in vivo. The COL-HANF scaffold was biodegradable and promoted bone regeneration eight weeks after the operation. Hence, we concluded that the COL-HANF scaffold has potential as a bone graft for bone tissue engineering.

Fabrication of polycaprolactone tubular scaffolds with an orthogonal-bilayer structure for smooth muscle cells.

In this study, we used electrospinning to prepare a bilayered polycaprolactone (PCL) tubular graft consisting of an internal layer comprising axial nanofibers and an external layer comprising circumferentially aligned nanofibers. Subsequently, the surfaces of the electrospun PCL tubular scaffolds were modified with 1,6-diaminohexane to introduce amino groups and were then chemically conjugated with gelatin (Gel). The amino groups and Gel were successfully immobilized on the PCL scaffolds according to a ninhydrin assay, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopic analysis and contact angle analysis. Additionally, vascular smooth muscle cells (vSMCs, A7r5) were cultured on random and aligned Gel-PCL scaffolds to evaluate the effects of fiber orientation on cell behavior. The results of immunofluorescence analysis showed that vSMCs on the aligned Gel-PCL scaffolds exhibited a pro-contractile phenotype.

Fabrication and Characteristics of PCL Membranes Containing Strontium-Substituted Hydroxyapatite Nanofibers for Guided Bone Regeneration.

Poly(ε-caprolactone) (PCL) membranes have been widely used in guided tissue regeneration (GTR) and guided bone regeneration (GBR). In addition, hydroxyapatite is the major inorganic component and an essential composition of hard bone and teeth. Recently, numerous studies have demonstrated that strontium-substituted hydroxyapatite (SrHA) not only enhances osteogenesis but also inhibits adipogenesis of mesenchymal stem cells. Therefore, SrHA incorporated into PCL could be an alternative material for GBR. In this study, strontium-substituted hydroxyapatite nanofibers (SrHANFs) were fabricated by a sol-gel route followed by electrospinning. We then fabricated PCL-SrHANF membranes as cell culture substrates and assessed the cellular behavior of osteoblast-like cells. Based on the observations of alkaline phosphatase (ALP) activity, bone sialoprotein (BSP) and osteocalcin (OCN) immunofluorescence staining, and Alizarin Red-S staining of cells cultured on the PCL-SrHANF and PCL membranes, we concluded that SrHANFs can promote the differentiation and mineralization of osteoblast-like cells and that PCL-SrHANF membranes have potential for GBR applications.

Beads of collagen-nanohydroxyapatite composites prepared by a biomimetic process and the effects of their surface texture on cellular behavior in MG63 osteoblast-like cells.

The aim of this work was to develop a novel method for preparing a three-dimensional bone-like matrix comprising nanohydroxyapatite crystals and fibrous collagen and to apply it for bone tissue engineering. Hydroxyapatite and collagen are the major components of natural hard bone. Therefore, they have been used extensively in orthopedic surgery as bone-filling materials. According to the principle of complex coacervation, three-dimensional collagen beads can be formed by extruding collagen solution into chondroitin sulfate A (CSA) solution. Subsequently, the collagen beads thus formed are soaked in simulated body-fluid solution to biomimic the formation process of natural bone matrix via the fabrication of collagen-nanohydroxyapatite beads. We also investigate the effect of the collagen-nanohydroxyapatite matrix on the proliferation and differentiation of MG63 cells. The presence of crystalline hydroxyapatite structure on the surface of fibrous collagen was confirmed by X-ray diffraction. MG63 cells cultured on the collagen-nanohydroxyapatite beads proliferate at the normal rate. Moreover, alkaline phosphatase (ALP) activity and the expression levels of three osteogenic genes, namely, type I collagen osteopontin and osteocalcin, in MG63 cells were significantly higher when the cells were cultured on collagen-nanohydroxyapatite beads than when they were cultured on collagen alone. The results of this study reveal that, in the presence of nanohydroxyapatite, the three-dimensional cell beads not only provide a substrate for cell growth but could also enhance the osteoblast-like cell differentiation of MG63 cells.

Compound Cellular Imaging of Laser Scanning Confocal Microscopy by Using Gold Nanoparticles and Dyes.

Combining the scattered light of gold nanoparticles (GNPs) and the fluorescence of dye molecules, a compound cellular imaging of laser scanning confocal microscopy (LSCM) is obtained. The human breast cancer cell line (MDA-MB-435S, BCRC 60429) is used for experiment. These cells are incubated with a glucose medium containing GNPs for 26 hours, and then are stained by Prodium Iodide (PI) for their nuclei. By using a single laser to illuminate these cells and adjusting the ranges of two bandpass filters for the detection, the scattered light from the GNPs and the fluorescence of PI can be induced simultaneously, but be detected separately without crosstalk. Furthermore, a compound cellular image can be obtained by merging the two images of the expressions of GNP and PI together. From the TEM images of these cells, it is observed that GNPs are aggregated in the vesicles of the cytoplasm due to the cell's endocytosis. The aggregation of GNPs makes the surface plasmon resonance band of GNPs broadened, so that strong scattered light from GNPs can be generated by the illumination of different-wavelength lasers (458, 488, 514, 561, and 633 nm).